Epstein-Barr Virus (EBV) is an ubiquitous human herpes virus that was first discovered in association with the African (endemic or e) form of Burkitt's lymphoma (BL). Subsequently the virus was also found associated with nasopharyngeal carcinoma (NPC) and was shown to be the causative agent of infectious mononucleosis (IM). Infection usually occurs during early childhood, generally resulting in a subclinical manifestation, occasionally with mild symptoms. Infection during adolescence or adulthood, however, can give rise to IM characterized by the presence of atypical lymphocytes in the periphery. The bulk of these lymphocytes are T lymphocytes; however, included in their number are a small population of B lymphocytes infected by EBV. The infection of B lymphocytes may also be accomplished in vitro. Such cells become transformed and proliferate indefinitely in culture and have been referred to as “immortalized”, “latently infected” or “growth transformed”. As far as is known, all individuals who become infected with EBV remain latently infected for life. This is reflected by the lifelong continuous presence of small numbers of EBV-genome positive transformed B-cells among the circulating peripheral blood lymphocytes and the continuous but periodic shedding of virus In the oropharynx.
In the vast majority of cases EBV infection results in a lynmphoproliferative disease that may be temporarily debilitating, but is always benign and self-limiting. In certain immunosuppressed individuals, however, the result can be uncontrolled lymphoproliferation leading to full-blown malignancy. This occurs in individuals who are immuno-suppressed intentionally, particularly children receiving organ transplants who are treated with cyclosporine A, or opportunistically, as in the case with individuals infected with HIV, or genetically, as in the case of affected males carrying the XLP (x-linked lymphoproliferative syndrome) gene. In these cases the resulting malignancies derive from the polyclonal proliferation of EBV-infected B cells. In addition, in such patients uncontrolled epithelial replication of the virus is detectable in lesions of oral hairy leukoplakia. Thus, the immune response plays a central role in the control of EBV infection.
Epstein Barr virus gene expression and molecular diagnostic approaches.
For many years Burkitt's lymphoma (BL) derived cell lines and EBV-transformed peripheral blood B-cells, also named lymphoblastoid cell lines (LCL) were considered to be the prototype model system for studying EBV-mediated transformation and oncogenesis.
During the last few years the entire DNA sequence of prototype virus strain, B95-8, has been determined. Analysis of this sequence has resulted in the identification of more than 80 open reading frames (Baer et al., Nature 310; 207-211 (1984)). The nomenclature for EBV reading frames is based on their position in the virus genome. The names begins with the initials of the BamH1 or EcoR1 restriction fragment where expression begins. The third character in the name if L or R, depending on whether the expression is leftward or rightward on the standard map. (so BLLF2 is the second leftward reading frame starting in BamH1 restriction fragment L.).
Basically three different gene transcription patterns have been observed in the various EBV-associated malignancies. These patterns are called latency type I, type II and type III, although recent data show the presence of additional transcripts complicating this typing system. Latency type I is characterized by the expression of Epstein Barr Nuclear Antigen 1 (EBNA-1; BKRF1) and the small non-coding RNA's Epstein Barr Early RNA 1 and 2 (EBER-1 and EBER-2). More recently a novel set of transcripts (BAFR0), with potential protein coding capacity in a number of small open reading frames included within these transcripts, has been found in all cells expressing the latency type I pattern. Latency type II is characterised by the expression of Latent Membrane Protein 1 (LMP-1; BNLF1) and LMP-2A/-2B (BNRF1), in addition to the type I transcripts mentioned above. LMP2 transcripts can only be expressed when the viral genome is in the covalently dosed circular form as these transcripts cross the terminal repeats on the viral genome and cannot be formed when the viral genome is in its linear “lytic” state. Latency type III is characterised by the expression of the nuclear antigens EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-4 (also referred to as EBNA-2, -3, -4, -6 and -5 respectively), in addition to the type II program transcripts. The expression of the different latency-associated transcription programs is influenced by host cell parameters, such as the level of methylation and cellular differentiation. As a consequence, EBV-gene expression can be observed to initiate from different promoter sites, depending upon the methylation state of the viral genome.
The association of expression of different latency type viral transcription profiles with the various EBV-associated malignancies has been determined in recent years mainly by means of Reverse-Transcripase Polymerase Chain Reaction (RT-PCR) analysis of RNA derived from tumor biopsy specimens or by analysis of cDNA libraries made from polyadenylated mRNA, selectively isolated from tumor tissue or in vitro (including in xenografted (nude) mice) propagated tumor cell lines and LCL's. Using these types of analysis, type I latency is found in BL tumor cells in vivo and sporadic BL cell lines in vitro. Type II latency is found in NPC, EBV-positive cases of Hodgkin's Lymphoma, T-cell, NK-cell and sporadic B-cell non-Hodgkin lymphoma (T-/NK-/B-NHL) and thymic and parotid carcinomas in the immunocompetent host, whereas type III latency patterns are found in most BL and LCL lines maintained in vitro and in pre-malignant lymphoproliferations and immunoblastic lymphoma which are observed mainly in immunocompromised individuals. In the latter populations sporadic lyomyosarcoma is also found which may express the type II pattern, whereas gastric carcinomas in non-compromised patients were found to express rather a type I latency pattern. There is sill no consensus on the exact transcription pattern of the truly latently infected B-cell that can be detected in the healthy EBV carrier. Depending on the method used for isolation of these latently infected B-cells, EBNA-1, EBER's and LMP-2 transcripts have been found, but also patterns including only EBNA-1 or only EBER's plus LMP-2 have been described.
It should be realised that these different patterns of viral (latent) gene transcription in B-cells and tumor tissue actually represents transcription in “bulk” (tumor) material and do not necessary reflect the expression pattern of each individual (tumor) cell. By immunohistochemical (IH) analysis of thin sections of various EBV-associated tumors using monoclonal antibodies to defined EBV latency-associated gene products, such as EBNA-1, EBNA-2 and LMP-1, a different picture is emerging. In recent studies using IH methodology it was found that the majority of tumor cells in AIDS and in post-transplant associated immunoblastic lymphoma display a pattern consistent with latency type I (only EBER-1/-2 and EBNA-1 detectable), whereas a minority express either latency type II (EBNA-1 plus LMP-1) or a novel form of latency characterised by the co-expression of EBNA-1 and EBNA-2 (Oudejans et al., Am. J. Pathol. 147 (1995) 923-933). Only rarely cells were observed that co-express EBNA-2 and LMP-1 which would be indicative for Latency type III. This could mean that the classic picture of viral gene expression associated with the different EBV-linked malignant diseases has to be revised to incorporate these more detailed findings.
In fact an even more differentiated picture is emerging as dearly different EBV-encoded genes are found to be expressed in different EBV-associated malignancies. Occasionally, viral gene products previously considered to belong to the (early) lytic phase of the viral life cycle are detectable, probably derived from occasional tumor cells switching to lytic viral replication under influence of local influences. This phenomenon can be clearly observed in nasopharyngeal carcinoma (NPC) where the switch to lytic replication in small nests of tumor cells is associated with cellular differentiation as revealed by the formation of cytokeratin filaments. Alternatively, such lytic gene products may derive from tumor infiltrating and differentiating B-cells carrying latent viral genomes, or from local endothelial and specialized epithelial cells that may become productively infected by EBV.
In addition to the latency-associated gene products and gene-expression clearly linked to local lytic viral replication, some viral genes usually considered to belong to the set of EBV early genes have been found to be expressed in selected EBV-associated tumors. These genes include viral homologues of cellular genes that may have a function in the pathogenesis of certain EBV-malignancies; e.g. BHRF1, the human Bcl-2 homologue providing apoptosis resistance and found to be expressed almost exclusively in B-NHL, or BARF1, a homologue of cellular ICAM-1, expressed in NPC and OHL but not in HD and other lymphoma, or BCRF1, the viral homologue of human IL-10 which may confer local immunomodulating activity mainly found in immunoblastic lymphoma in immunocompromised patients, or BDLF2, which has some homology to cellular cydin B1 and may function on overriding normal cell cycle control.
Furthermore, genes that effectively mediate the switch from latent to lytic cycle gene expression in vitro can be found to be expressed in vivo without detectable full lytic cycle induction, a situation referred to as restricted or abortive lytic gene expression.
Therefore, at the single cell level, EBV gene expression is not homogeneously distributed throughout the tumor and different tumor cell populations may express (slightly) different patterns of EBV genes. Thus, in addition to analysing EBV gene expression in nucleic acid extracts prepared from whole tumor biopsy samples, information on viral gene expression at the single cell level is required to accurately describe the transcriptional activity of the EBV genome in the tumor cells.
It has been suggested that the switch to lytic gene expression may be positively related to success of therapy, as such cells are less resistant to apoptosis and are more immunogenic, thus being more sensitive to drug/radiation therapy and host (immune) surveillance and repair mechanisms. Thus, in addition to analysing the latency associated gene transcripts, accurate detection and relative quantitation of EBV-encoded viral lytic gene products in the tumor is of diagnostic and prognostic relevance.
In addition to its use in specific diagnosis and monitoring of the EBV-associated malignancies as described above, analysis of viral gene expression may be of relevance in differential diagnosis of oral hairy leukoplakia, which is characterised by expression of viral lytic genes in the absence of detectable EBNA-1 and EBER expression and for diagnosing acute and chronic/persistent B-cell lymphoproliferations which may have a self-limiting or non-malignant progression.
All these findings point to the relevance of accurate determination of type and level of viral gene expression for diagnosis of EBV-associated malignancies and pre-malignant lymphoproliferations.
In addition to or instead of analysis of viral gene expression in tumor or otherwise affected tissue specimens, detection and quantitation of virus infected (tumor) cells in the circulation and analysis of viral gene expression in these cells may provide a more accessible means of molecular diagnosis, not only applicable for detection of circulating tumor cells in already affected patients or for pre-emptive screening purposes in patients at risk, such as post-transplant- and AIDS-patients and otherwise immuno-compromised individuals, but also relevant for monitoring the effect(s) of anti-tumor therapy.
Besides measuring of the EBV-associated tumor load, which may be achieved by quantitating the level of viral DNA in a particular patient specimen, the qualitative and quantitative analysis of viral gene transcription is essential for differential diagnosis and prognosis and may be relevant for determining therapeutic intervention strategies.
Molecular analysis using either nucleic acid or immunologic reagents requires detailed knowledge of the target molecules involved, especially regarding strain/epitope variation. Selection of gene segments and epitopes that are highly conserved among different EBV-strains and isolates is of crucial importance for design and development of diagnostic reagents that can be applied to world-wide clinical diagnosis as indicated above. On the other hand analysis of mutations, deletions or insertions into specific viral gene products leading to expression of proteins with potential modified function may be of value for epidemiological and pathogenic studies and may have potential diagnostic relevance. For example EBV strain variation can be determined by analysing the sequence of especially the Epstein Barr Nuclear Antigen (EBNA)-2 and -3 genes, which contain specific sequences that allows differentiation into EBV strain types A and B, the B-strain being relatively more frequent in AIDS-associated lymphoma and in certain parts of the world. On the other hand, sequence variations (esp. point mutations and deletions) have been described for the EBNA-1, Latent Membrane Protein (LMP)-1, LMP-2 and ZEBRA encoding genes, of which the LMP-1 specific 30 bp deletion variant has been linked to a more aggressive oncogenic phenotype.
The availability of techniques to specifically analyse viral DNA and expressed RNA and protein are required for accurate diagnosis. One example of a technique for the amplification of a DNA target segment is the so-called “polymerase chain reaction” (PCR). PCR in combination with the proper primer sets is well suited for detection of viral DNA, whereas immunohistochemistry combined with appropriate antibody reagents is the method of choice for visualization of tumor associated viral proteins. High copy numbers of viral RNA can be detected by RNA in situ hybridization as routinely applied for the detection of EBER-1 and -2, which are expressed at extremely high copy numbers in virtually all EBV-associated tumors. The detection of low copy numbers of viral mRNA requires more sensitive techniques such as RT-PCR and Nucleic Acid Sequence Based Amplification (NASBA). Application of RT-PCR is seriously hampered by the need for spliced mRNA in order to allow viral gene expression in a viral DNA background therefore limiting its use to only a selected set of spliced viral genes. In addition, the need for high temperatures in the PCR part of the RT-PCR reaction seriously limits its application to in situ diagnostic approaches.
Another drawback of RT-PCR is the requirement of splice sites within the transcript of interest to exclude amplification of genomic DNA and the fact that it is a two-step reaction.
These limitations are overcome by using the NASBA approach for analysing viral mRNA expression both in tissue extracts and by in situ analysis at the single cell level. NASBA allows selective amplification of reading frame or exon-specific viral mRNA in a viral DNA background and allows visualizabon of (viral) mRNA expression in thin sections of tumor tissue without affecting cell morphology (in situ NASBA). As NASBA is not limited by the need for choosing specific primer sets spanning intron sequences, exon-specific primers and probes may be utilized. NASBA also allows more simple and broadly applicable analysis of genetic variations in expressed viral genes. Using NASBA, RNA but not genomic DNA is amplified independently of splice sites.
Based on their splicing patterns, four types of EBV transcripts can be distinguished:    Transcripts which are extensively spliced in the noncoding region but not in the coding region, like EBNA1 transcripts (Kerr et al., Virol; 187:189-201 (1992)).    Transcripts which are spliced in the coding domain, like LMP1 and LMP2 (Laux et al., J Gen Virol: 70: 3079-84 (1989)).    Transcripts which are not spliced at all, like the EBER1 and EBER2 transcripts (Clemens, Mol Biol Reports; 17: 81-92 (1993)).    Transcripts of which splicing patterns are not known. These are merely “early” transcripts, like BARF1 (Zhang et al., J Virol; 62(2):1862-9 (1988)), BDLF2 and BCRF1 (Vieira et al., PNAS; 88(4):1172-6 (1991)).
The present invention is related to the detection of a certain EBV mRNAs and provides oligonucleotides suitable for use in the amplification and subsequent detection of these mRNAs. The binding sites of the oligonucleotides according to the present invention are located in the following EBV genes:    Epstein Barr Early RNA 1 (EBER-1), Epstein Barr Nuclear Antigen 1 ( EBNA-1), Latent Membrane Protein 1 (LMP-1), LMP-2, and vIL10 (BCRF-1). BARF1, and BDLF2 (all characterised by the nomenclature of Baer et al., Nature. vol., 310, pp 207-211, 1984).
An embodiment of the present invention is directed to oligonucleotides which are 10-35 nucleotides in length and comprise, at least a fragment of 10 nucleotides, of a sequence selected from the group consisting of:    EBNA-1, [the BKRF1reading frame spanning nucleotides 107950-109872],    EBER-1, [reading frame spanning nucleotides 6629-6795],    LMP-1, [the BNLF1 reading frame spanning nucleotides 169474-169207],    LMP-2, [exons 2, 3, 4, 5, 6, 7 and 8 spanning nucleotides 58-272, 360-458, 540-788, 871-951, 1026-1196, 1280-1495 and 1574-1682 respectively],    vIL10, [BCRF1 reading frame spanning nucleotides 8675-101841],    BARF1, [the reading frame spanning nucleotides 165504-166166], or    BDLF2, [the reading frame spanning nucleotides 132389-131130],wherein all reading frame spanning nucleotide numbers are according to Baer et al., 1984.
Preferred oligonucleotides according to the present invention are 10-35 nucleotides in length and comprise, at least a fragment of 10 nucleotides, of a sequence selected from the group consisting of:    1.1, 5′-GCCGGTGTGTTGTTCGTATATGG-3′ [SEQ.ID.NO.: 1],    1.2, 5′-CTCCCTTTACAACCTAAGGC-3′ [SEQ.ID.NO.: 2],    2.1, 5′-AGAGACAAGGTCCTTAATCGCATCC-3′ [SEQ.ID.NO.: 3], or    2.2, 5′-AATACAGACAATGGACTCCC-3′ [SEQ.ID.NO.: 4], or its complementary sequence (EBNA-1), or    1.1, 5′-CGGGCGGACCAGCTGTACTTGA-3′ [SEQ.ID.NO.: 6],    2.2, 5′-GAGGTTTTGATAGGGAGAGGAGA-3′ [SEQ.ID.NO.: 7],    54, 5′-CGGACCACCAGCTGGTACTTGA-3′ [SEQ.ID.NO.: 8],    55, 55′-GCTGCCCTAGAGGGTTTTGCTA-3′ [SEQ.ID.NO.: 9], or    56, 5′-CGAGACGGCAGAAAGCAGA-3′ [SEQ.ID.NO.: 10], or its complementary sequence (EBER-1), or    1.1, 5′-ATACCTAAGACAAGTTTGCT-3′ [SEQ.ID.NO.: 12],    1.2, 5′-ATCAACCAATAGAGTCCACCA-3′ [SEQ.ID.NO.: 13],    2.1, 5′-CATCGTTATGAGTGACTGGA-3′ [SEQ.ID.NO.: 14], or    2.2, 5′-ACTGATGATCACCCTCCTGCTCA-3′ [SEQ.ID.NO.: 15], or its complementary sequence (LMP-1), or    1.1, 5′-TAACTGTGGTTTCCATGACG-3′′ [SEQ.ID.NO.: 17],    1.2, 5′-AGGTACTCTTGGTGCAGCCC-3′ [SEQ.ID.NO.: 18],    2.1, 5′-AGCATATAGGAACAGTCGTGCC-3′ [SEQ.ID.NO.: 19], or    2.2, 5′-AGTGGACATGAAGAGCACGAA-3′ [SEQ.ID.NO.: 20], or its complementary sequence (LMP-2), or    1.1, 5′-CAGGTTCATCGCTCAGCTCC-3′ [SEQ.ID.NO.: 22],    1.2, 5′-GGCTGTCACCGCTTTCTTGG-3′ [SEQ.ID.NO.: 23],    2.1, 5′-AGTGTTGGCACTTCTGTGG-3′ [SEQ.ID.NO.: 24], or     2.2, 5′-AGCATGGGAGATGTTGGCAGC-3′ [SEQ.ID.NO.: 25], or its complementary sequence (BARF-1), or    1.1, 5′-TGGAGCGAAGGTTAGTGGTC-3′ [SEQ.ID.NO.: 27],    1.2, 5′-TACCTGGCACCTGAGTGTGGAG-3′ [SEQ.ID.NO.: 28],    2.1, 5′-AGAATTGGATCATTTCTGACAGGG-3′ [SEQ.ID.NO.: 29], or    2.2, 5′-AGACATGGTCTTTGGCTTCAGGGTC-3′ [SEQ.ID.NO.: 30], or its complementary sequence (vIL10 (BCRF1)), or    1.1, 5′-CTACCTTCCACGACTTCACC-3′ [SEQ.ID.NO.: 32],    1.2, 5′-AAGTCTTTTATAAGGCTCCGGC-3′ [SEQ.ID.NO.: 33],    2.1, 5′-AGGCCATGGTGTCATCCATC3′ [SEQ.ID.NO.: 34], or    2.2, 5′-AGAGAGAGAGTAGGTCCGCGG-3′ [SEQ.ID.NO.: 35], or its complementary sequence (BDLF2).
A preferred embodiment of the present invention is directed to an oligonucleotide linked to a suitable promoter sequence.
A more preferred embodiment of the present invention is directed to a pair of oligonucleotides, for the amplification of a target sequence within a Epstein Barr virus sequence, for use as a set, comprising:    1.2, 5′-CTCCCTTTACAACCTAAGGC-3′ [SEQ.ID.NO.: 2], and    2.1, 5′-AGAGACAAGGTCCTTAATCGCATCC-3′ [SEQ.ID.NO.: 3] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′[SEQ.ID.NO.: 37] (EBNA-1); or    1.1, 5′-CGGGCGGACCAGCTGTACTTGA-3′ [SEQ.ID.NO.: 6] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′[SEQ.ID.NO.: 37], and    2.2, 5′-GAGGTTTTGATAGGGAGAGGAGA-3′ [SEQ.ID.NO.: 7] (EBER-1);    1.1, 5′-ATACCTAAGACAAGTTTGCT-3′ [SEQ.ID.NO.: 12] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′[SEQ.ID.NO.: 37], and    2.1, 5′-CATCGTTATGAGTGACTGGA-3′ [SEQ.ID.NO.: 14] (LMP-1); or    1.2, 5′-aggtactcttggtgcagccc-3′ [SEQ.ID.NO.: 18], and    2.1, 5′-agcatataggaacagtcgtgcc-3′ [SEQ.ID.NO.: 19] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′ [SEQ.ID.NO.: 37] (LMP-2); or    1.2, 5′-ggctgtcaccgctttcttgg-3′ [SEQ.ID.NO.: 23], and    2.1, 5′-agtgttggcacttctgtgg-3′ [SEQ.ID.NO.: 24] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′ [SEQ.ID.NO.: 37] (BARF-1); or    1.1, 5′-TGGAGCGAAGGTTAGTGGTC-3′ [SEQ.ID.NO.: 27], and    2.2, 5′-AGACATGGTCTTTGGCTTCAGGGTC-3′ [SEQ.ID.NO.: 30] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′[SEQ.ID.NO.: 37] (vIL10 (BCRF1)); or    1.1, 5′-CTACCTTCCACGACTTCACC-3′ [SEQ.ID.NO.: 32] provided with a T7 polymerase promoter sequence 5′-aattctaatacgactcactataggg-3′ [SEQ. ID.NO.: 37] and    2.1, 5′-AGGCCATGGTGTCATCCATC-3′ [SEQ.ID.NO.: 34], or    2.2, 5′-AGAGAGAGAGTAGGTCCGCGG-3′ [SEQ.ID.NO.: 35] (BDLF2).
The term “oligonucleotide” as used herein refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides such as primers and probes.
The term “primer” as used herein refers to an oligonucleotide either naturally occurring (e.g. as a restriction fragment) or produced synthetically, which is capable of acting as a point of initiation of synthesis of a primer extension product which is complementary to a nucleic acid strand (template or target sequence) when placed under suitable conditions (e.g. buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization, such as DNA dependent or RNA dependent polymerase. A primer must be sufficiently long to prime the synthesis of extension products in the presence of an agent for polymerization. A typical primer contains at least about 10 nucleotides in length of a sequence substantially complementary (P1) or homologous (P2) to the target sequence, but somewhat longer primers are preferred. Usually primers contain about 15-26 nucleotides but longer primers may also be employed.
Normally a set of primers will consist of at least two primers, one ‘upstream’ and one ‘downstream’ primer which together define the amplificate (the sequence that will be amplified using said primers).
The oligonucleotides according to the invention may also be linked to a promoter sequence. The term “promoter sequence” defines a region of a nucleic add sequence that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the process of transcription by which an RNA transcript is produced. In principle any promoter sequence may be employed for which there is a known and available polymerase that is capable of recognizing the initiation sequence. Known and useful promoters are those that are recognized by certain bacteriophage RNA polymerases such as bacteriophage T3, T7 or SP6.
It is understood that oligonucleotides consisting of the sequences of the present invention may contain minor deletions, additions and/or substitutions of nucleic acid bases, to the extent that such alterations do not negatively affect the yield or product obtained to a significant degree.
Another preferred embodiment of the present invention is directed to an oligonucleotides which are 10-35 nucleotides in length and comprise, at least a fragment of 10 nucleotides, of a sequence selected from the group consisting of:    5′-CGTCTCCCCTTTGGAATGGCCCCTGGACCC-3′ [SEQ.ID.NO.: 5] (EBNA-1),    5′-GTACAAGTCCCGGGTGGTGAG-3′ [SEQ.ID.NO.: 11] (EBER-1),    5′-GGACAGGCATTGTTCCTTGG-3′ [SEQ.ID.NO.: 16] (LMP-1),    5′-AGCTCTGGCACTGCTAGCGTCACTGATTTT-3′ [SEQ.ID.NO.: 21] (LMP-2),    5′-CTGGTTTAAACTGGGCCCAGGAGAGGAGCA-3′ [SEQ.ID.NO.: 26] (BARF-1),    5′-CAGACCAATGTGACAATTTTCCCCAAATGT-3′ [SEQ.ID.NO.: 31] (vIL10 (BCRF1)), or    5′-CCAATGGGGGAGGAGAGACCAAGACCAATA-3′ [SEQ.ID.NO.: 36] (BDLF2),provided with a detectable label. Said oligonucleotides may be used for the detection of the amplificate generated using the oligonucleotides according to the present invention. Probes comprising said sequence are also part of the present invention.
An oligonucleotide sequence used as detection-probe may be labeled with a detectable moiety. Various labeling moieties are known in the art. Said moiety may, for example, either be a radioactive compound, a detectable enzyme (e.g. horse radish peroxidase (HRP)) or any other moiety capable of generating a detectable signal such as a colorimetric, fluorescent, chemiluminescent or electrochemiluminescent signal. Preferred analysis systems wherein said labels are used are electrochemiluminescence (ECL) based analysis or enzyme linked gel assay (ELGA) based analysis.
Another preferred embodiment of the present invention is directed to a method for the detection of EBV-specific RNA sequences in human tissue (extracts), peripheral blood and white blood cells, body fluids, tumor cell lines, etc. using the oligonucleotides according to the present invention. Said method comprising the following steps:                amplifying a target sequence within said mRNA using (a pair of oligonucleotides according to the invention and suitable amplification reagents,        reacting the sample, optionally containing amplified nucleic acid, with an oligonucleotide according to the present invention as a detection-probe,        detecting hybrids formed between the amplified sequence and the probe.        
Various techniques for amplifying nucleic acid are known in the art. One example of a technique for the amplification of a DNA target segment is the so-called “polymerase chain reaction” (PCR). With the PCR technique the copy number of a particular target segment is increased exponentially with a number of cycles. A pair of primers is used and in each cycle a DNA primer is annealed to the 3′ side of each of the two strands of the double stranded DNA-target sequence. The primers are extended with a DNA polymerase in the presence of the various mononucleotides to generate double stranded DNA again. The strands of the double stranded DNA are separated from each other by thermal denaturation and each strand serves as a template for primer annealing and subsequent elongation in a following cycle. The PCR method has been described in Saiki et al., Science 230, 135, 1985 and in European Patents no. EP 200362 and EP 201184.
Another technique for the amplification of nucleic acid is the so-called transcription based amplification system (TAS). The TAS method is described in International Patent Appl. no. WO 88/10315. Transcription based amplification techniques usually comprise treating target nucleic acid with two oligonucleotides one of which comprises a promoter sequence, to generate a template including a functional promoter. Multiple copies of RNA are transcribed form said template and can serve as a basis for further amplification.
An isothermal continuous transcription based amplification method is the so-called NASBA process (“NASBA”) as described in European Patent no. EP 329822. NASBA includes the use of T7 RNA polymerase to transcribe multiple copies of RNA from a template including a T7 promoter.
For RNA amplification (as with the method according to the invention), the NASBA technology, or another transcription based amplification technique, is a preferred technology. If RT-PCR is used for the detection of viral transcripts differentiation of mRNA- and DNA-derived PCR products is necessary. For spliced transcripts, like the IEA mRNA, the exonintron structure can be used. However, mRNA species encoding the late structural proteins are almost elusively encoded by unspliced transcripts. DNAse treatment prior to RT-PCR can be employed (Bitsch, A. et al., J Infect. Dis 167, 740-743, 1993; Meyer, T. et al., Mol. Cell Probes. 8, 261-271, 1994), but sometimes fails to remove contaminating DNA sufficiently (Bitsch, A. et al., 1993).
In contrast to RT-PCR, NASBA, which is based on RNA transcription by T7 RNA polymerase (Kievits et al., J Virol Meth; 35:273-86), does not need differentiation between RNA- and DNA-derived amplification products since it only uses RNA as its principal target. NASBA enables specific amplification of RNA targets even in a background of DNA.
This method was used for the analysis of EBV transcripts in whole blood samples from HIV-infected individuals.
Test kits for the detection of EBV in clinical samples are also part of the present invention. A test kit according to the invention may comprise a pair of oligonucleotides according to the invention and a probe comprising an oligonucleotide according to the invention. Such a test kit may additionally comprise suitable amplification reagents such as DNA and or RNA polymerases and mononucleotides. Test kits that can be used with the method according to the invention may comprise the oligonucleotides according to the invention for the amplification and subsequent detection of EBV-specific RNA sequences.
The invention is further exemplified by the following examples.